1. Field of the Invention
The invention relates immunoassay methods for detecting antibodies specific for oxidized DNA bases. Such methods are useful in the diagnosis and monitoring of inflammatory and autoimmune diseases.
2. Description of the Background Art
Chronic inflammation is known to be involved in a large number of diseases, including autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) as well as in cancer (J. C. Fantone et al., Am. J. Pathol. 07, 397 (1982); B. A. Freeman et al., Lab Invest. 47, 412 (1982); Y. Niwa et al., Inflammation 9, 163 (1985); C. E. Cross et al., Ann, Int, Med, 107, 526 (1987); S. Blount et al., Clin. Exp. Immunol, 81, 384 (1990)). In SLE, autoantibodies are produced which are directed against an array of nuclear antigens. Such antibodies, known as antinuclear antibodies or ANA, include antibodies reactive with the patient's own DNA. The ultimate stimulus for production of ANA and anti-DNA antibodies in these patients is not clear (B. D. Stollar, Clinics in Immunology and Allergy 1(2), 243 (1981); A. J. G. Swaak et al., Ann. Rheum. Dis. 40, 45 (1981); E. M. Tan et al., Arthritis Rheum. 25 (11.), 1271 (1982); T. Swaak et al., Ann. Rheum. Dis. 44, 245 (1985); D. S. Pisetsky et. al., Arthritis Rheum. 33(2), 153 (1990)).
Of the various serological markers, the presence of circulating antibodies reactive with double-stranded (ds) DNA seems to best correlate with the clinical manifestations of SLE. Complexes of DNA with anti-dsDNA antibodies are thought to precipitate in the renal glomeruli and result in glomerulo-nephritis (C. Bruneau et al., J. clin. Invest. 64, 191 (1979)). However, the factor or factors responsible for the release of DNA fragments from nuclear DNA is not yet known.
Oxidative stress contributes to the pathogenesis of a broad array of diseases, including autoimmune diseases, as described above, cardiac disease such as ischemia/reperfusion injury (Jolly, S. R. et al., Circ. Res. 34:277-285 (1984); McCord, J. M. New Engl. J, Med. 312:159-163 (1985), and neoplastic abnormalities (Vuillaume, M., Murat, Res. 1.86:43-72 (1987); Malins, D.C. et al., Canc. Res. 51:5430-5432 (1991)). Inflammatory conditions are associated with increased infiltration of phagocytic cells into reactive tissue sites. This infiltration is also accompanied by enhanced oxidant formation (J. A. Badewy et al., Annu. Rev. Biochem. 49, 695 (1980); S. J. Klebanoff, Ann. Intern. Med. 93, 480 (1980); B. M. Babior, Blood 64, 959 (1984); S. A. Weitzman et al., Blood 76, 655 (1990); P. Biemond et al., J. Clin. 73, 1576 (1984)). Phagocytic cells, in particular polymorphonuclear leukocytes (PMNs) or neutrophils, generate large amounts of active oxygen species when activated by a variety of stimuli including immune complexes or complement-derived fragments (Fantone et al., supra; Freeman et al., supra; Niwa et al., supra; Cross et al., supra; Badewy et al., supra; Klebanoff, supra; Babior, supra; Weitzman et al., supra; K. Frenkel, Environ, Health Persp. 81, 45 (1989); K. T. Oldham et al., Free Rad. Biol. Med. 4, 387 (1988)). Chronic inflammation increases the incidence of autoimmune disease and cancer (Cross et al., supra; vuillaume, supra). ulcerative coliris may progress to colon cancer, while pulmonary infiltration and activation of PMNs and alveolar macrophages are factors in the pathogenesis of lung cancer (Cross et al., supra).
The reactive oxygen species include superoxide anion radicals that dismutate either spontaneously or enzymatically to hydrogen peroxide (H.sub.2 O.sub.2) (K. Frenkel, supra). Peroxide has been shown to activate the complement pathway in the presence of Fe or Cu ions (M. Shingu et al., Dermatologica 179 (Suppl. 1), 107 (1989)). Furthermore, it is H.sub.2 O.sub.2 that traverses cellular and nuclear membranes almost like water, and reaches the nuclear DNA (K. Frenkel, supra; R. Meneghini, Mutation Res. 195, 215 (1988); B. Halliwell et al., Arch. Biochip. Biophys. 246, 501 (1986); M. Chevion, Free Rad. Biol. Med. 5, 27 (1988)). Once in the nucleus, H.sub.2 O.sub.2 reacts at sites that contain bound Fe or Cu, leading to the formation of hydroxyl radical (.OH)-like species that cause site-specific damage to DNA. Indeed, H.sub.2 O.sub.2 generated by a number of cellular processes is known to cause DNA strand breaks and to oxidize DNA bases. Products of this oxidation include 5-hydroxymethyl uracil (HMU), thymine glycol (TG) and 8-hydroxyguanine (8-OHG) (Frenkel, supra; D. R. Dutton et al., Carcinogenesis 6:1279 (1985); K. Frenkel et al., Cancer Res, 46, 5533 (1986); K. Frenkel et al., Carcinogenesis 8, 455 (1987); K. Frenkel et al., in: Oxy-Radicals in Molecular Biology and Pathology, P. A. Cerutti et al., Eds., Alan R. Liss, Inc., New York, N.Y., 1988, pp. 509-524; K. Frenkel et al., Free Rad. Biol. Med. 9(Suppl. 1), 170 (1990); H. Kasai et al., Carcinogenesis 7, 1849 (1986); E. S. Fiala et al., Cancer Res. 49, 5518 (1989); S. A. Leadon, Brit. J. Cancer 55(Suppl. 8), 113 (1987); J. G. Lewis et al., Cancer Res. 45:1270 (1985); J. H. Jackson et. al., J. Clin Invest. 84, 1644 (1989); K. S. Kasprzak et al., Carcinogenesis. 11, 647 (1990)).
Sera of SLE patients may contain increased amounts of catalase (M. Shingu et al., supra); this enhancement is characteristic of oxidative stress (G. Storz et al., TIG 6(11), 363 (1990)).
Rabbit polyclonal antibodies specific for the oxidized base thymine glycol have been described (Rajagopalan et., Radiat. Res. 97:499-510 (1984)). Monoclonal antibodies specific for thymine glycol have been produced and used to detect thymine glycol in oxidized DNA by ELISA (Leadon, S. A. et al., Mutat. Res. 112:191-200 (1983); Kaneko, M. et al., Canc, Res. 46:71-75 (1986); Leadon, S. A., Brit. J. Canc. 55 (Suppl VIII):113-167 (1987); Hubbard, K. et al., Radiat. Res. 118:257-268 (1989)). In all of these studies, the antibody was produced as a reagent for the purpose of identifying oxidative damage to DNA in immunoassays as an improvement over enzymatic and other biochemical assays to detect such alterations. Thus, the assays always involved whole DNA or polynucleotides as the analyte.
Because the previous work on thymine glycol antibodies was intended to provide an assay for the presence of the modified base in DNA, a useful assay in which the analyte is the antibody specific for an oxidized DNA base has not been previously contemplated and is not currently available. The ability to measure such antibodies would be of great value for early diagnosis of inflammatory conditions associated with oxidative damage, and for monitoring the progress of such conditions and their response to therapy. For example, Djuric et al. (J. Nat'l. Canc., Inst, 83:766-769 (1991)) found a positive correlation between fat intake and the presence of HMU in white blood cells of women at high risk for breast cancer. The methods and compositions of the present invention would be useful to monitor such high risk individuals for oxidative damage prior to the onset of frank neoplastic disease.
Caruthers et al. (U.S. Pat. No. 4,458,066) discloses nucleotides covalently bound to an inorganic polymer support, for the synthesis of oligonucleotide chains. Caruthers et al. does not suggest the use of nucleotides containing oxidized bases. Importantly, the methods disclosed would not be useful with oxidized bases because reactive groups (e.g., alcohols) on the oxidized base's ring structure would interfere in the covalent attachment via the sugar moiety to the inorganic polymer. Furthermore, Caruthers et al. does not suggest the use of nucleosides "artificially polymerized" by binding to a carrier such as a protein, wherein the carrier is then non-covalently attached to a solid support. Furthermore, Caruthers et al. only envisions inorganic supports.